Black widow spiders can inflict intense pain from potentially life-threatening bites, and are a leading cause of hospitalizations for severe envenomation. The extreme toxicity of black widow venom is due to a large family of unique neurotoxins (latrotoxins) that trigger massive exocytosis of neurotransmitters. Latrotoxins have a narrow phylogenetic distribution restricted to a single spider family, suggesting that black widow venom may be particularly toxic due to recent gene family expansion. Along with latrotoxins, black widow venom contains several other protein families that are likely to contribute to envenomation. Yet virtually nothing is known regarding the venoms of closely related less hazardous species that could identify key differences in venom protein composition that correlate with neurotoxicity or reveal the evolutionary processes that led to these differences. A major objective of this renewal project is to leverage newly available spider genomic resources to better understand the evolution of hazardous venoms and to test whether black widow venom is enriched with a diversity of unique toxins as a result of substantial lineage-specific molecular evolution. The project will build on a recently completed black widow transcriptome and the newly available genome of the closely related but less hazardous house spider, available through the i5k (5000 arthropod genomes) initiative. The genomic location, structure, and size of gene families that encode candidate venom proteins will be characterized from the house spider genome largely using bioinformatics approaches (Aim 1). Genome-informed proteomic analyses of venom using mass spectrometry and multi-tissue gene expression studies will test candidate venom gene predictions and reveal compositional differences between black widow and house spider venoms (Aim 2). Phylogenetic trees of functionally important protein families in black widow venom and their homologs from multiple spider transcriptomes and genomes will be estimated to identify changes in protein diversity and expression that correlate with venom toxicity (Aim 3). The work to accomplish each of these Aims will closely involve multiple undergraduate student researchers. This project will significantly advance fundamental understanding of venom evolution, with results having translational applications for drug discovery, the design of neurophysiological research tools and improved antivenoms. This project will also contribute significant resources to a public genome initiative.